Despite the progress made towards the development of microfabricated DNA analysis systems, there are significant hurdles to the realization of small, portable DNA analysis systems. Although the cross sectional dimensions of typical microfabricated electrophoresis channels are on the order of those employed in conventional capillary systems, scale-down efforts have proven to be less than straightforward. Most of the miniaturized separation systems developed so far have adopted non-crosslinked polymer solutions as the medium of choice for DNA separations. The use of non-cross-linked sieving media necessitates the use of high electric fields to overcome the diffusion/dispersion of DNA fragments in these media and hinders the use of battery operation. In addition, most of these devices require external optical readers thereby increasing the cost substantially. While laser induced fluorescence provides detection of even single molecules, having a detection system orders of magnitude larger than the separation systems significantly diminishes the benefits of miniaturization (Woolley, A. T., et al., Anal. Chem. 70:684–688, 1998). Finally, high-resolution separations tend to require long separation lengths that result in devices with a relatively large footprint. These are some of the limitations that have restricted the applicability of microfabricated devices to laboratory based settings.
Unless DNA separations can be performed over much shorter length scales, it will be difficult to realize the enormous cost savings possible through mass production via photolithographic fabrication techniques. These techniques, routinely employed in the semiconductor industry, have the potential to allow tens or hundreds of devices to be produced on a single wafer. For example, once initial design costs have been paid, fabrication of an integrated DNA analysis device incorporating on-chip temperature control and fluorescence detection costs about $5000 per 25 wafers (Burns, M. A., et al., Science 282:484–487, 1998). If the separation length can be reduced to 0.5 cm, a yield of approximately 120 devices can be expected from a 10 cm wafer, resulting in a net cost just over $1.50 per device. This represents an incredible cost savings compared to macroscale DNA analysis systems, and becomes even more dramatic when combined with the essentially negligible reagent costs associated with the nanoliter sample volumes required.
Polyacrylamide gel electrophoresis (PAGE) offers the advantage of requiring significantly less power to achieve efficient separations over shorter separation lengths than separations in non-crosslinked media. These characteristics polyacrylamide gels make it an attractive choice for the development of a low-power, gel-preloaded, DNA analysis system. However, the use of polyacrylamide gels in microfabricated devices has been hindered by the same problems that have plagued traditional slab gel and capillary gel electrophoresis systems (Schmalzing, D. et al., Electrophoresis 20:3066–3077, 1999). Problems associated with in-situ preparation (lack of precise control over and knowledge of chemical properties and purity of the resulting product), short shelf life, operation and sample injection still need to be resolved. The in-situ polymerization of polyacrylamide also does not offer stringent control on the position, and shape of the resulting gel interface, which defines the shape of the injected sample. Localization of the separation matrix to a pre-defined section of the device is necessary for the development of integrated analysis systems incorporating sample preparation steps prior to the separation stage (Burns, M. A., et al., Science 282:484–487, 1998).
Along with the proper matrix composition and gel interface shape, the application of the sample to the gel interface is an important consideration. In all microfabricated separation systems, the process of loading the sample onto the separation matrix is extremely critical in determining the performance of these devices. Several sample injector designs have been used, most notably the cross injector (Jacobson, S. C., et al. Anal. Chem. 66:1107–1113, 1994; Fan, Z. H. and D. J., Harrison, Anal. Chem. 66:177–184, 1994; Manz, A., et al., Trends Anal Chem. 10:144–149, 1991) and the double-T injector (Manz, A., et al., Trends Anal Chem. 10:144–149, 1991; Effenhauser, C. S., et al., Anal. Chem. 65:2637–2642, 1993) design. While these sample injection schemes allow a sample plug of defined volume to be injected (typically 50–500 pL), no significant increase in sample concentration is achieved. Hence, reducing the size of the sample injection plug-width also reduces the amount of sample injected. Independent control of the amount of sample injected is difficult, necessitating the use of highly sensitive detectors to detect the separated fragments.
Electrokinetic focusing techniques have been modified and used to achieve an increase in sample concentration in microfabricated devices by introducing a mismatch between the sample buffer and a focusing buffer (Jacobson, S. C. and J. M. Ramsey, Anal. Chem. 69:3212–3217, 1997). Similarly, sample stacking has been achieved by positioning a porous membrane positioned immediately upstream of the gel interface (Khandurina, J., et al., Anal. Chem. 71:1815–1819, 1999). Unfortunately, both these techniques require electric potentials on the order of 1 to 2 kV to operate effectively. More recently, a sample compaction mechanism based on entropic trapping has been demonstrated (Han, J. and H. G. Craighead, Science 288:1026–1029, 2000). The DNA is trapped entropically in a well at low electric field strengths and can be released from the well as a well-defined plug by increasing the electric field strengths. However, this type of injection has currently been shown to work only for relatively large DNA molecules.